PLGA substrate with aligned and nano-sized surface structures and associated method

ABSTRACT

A substrate for promoting growth of chondrocytes to repair articular cartilage is disclosed. The substrate comprises a polymeric material comprising aligned and nano-sized surface structures. An associated method is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/452,847 filed on Mar. 7, 2003 and claims priority as acontinuation-in-part to U.S. patent application Ser. No. 10/634,292filed on Aug. 5, 2003, the disclosures of which are hereby incorporatedby reference herein. U.S. patent application Ser. No. 10/634,292 claimspriority to U.S. Provisional Patent Application No. 60/401,060 which wasfiled on Aug. 5, 2002 and is hereby incorporated by reference herein.Cross reference is made to international application numberPCT/US02/25812 which was filed on Aug. 14, 2002, is hereby incorporatedby reference herein, and claims the benefit of U.S. Provisional PatentApplication No. 60/312,800 which was filed on Aug. 16, 2001 and ishereby incorporated by reference herein.

GOVERNMENT RIGHTS

Research relating to the present application was supported by the U.S.Government under National Science Foundation Grant No. DGE-99-72770. TheU.S. Government may have certain rights in this application.

FIELD

This application relates to substrates for promoting tissue growth.

BACKGROUND

Articular cartilage provides joints with excellent friction, coating,and wear properties necessary for knee movement, such as constantgliding. Articular cartilage consists of extracellular matrix (composedof collagen, poteoglycans, and water) and chondrocytes(cartilage-synthesizing cells). Collagen fibers in cartilage are alignedand generally have thin diameters, ranging from 10 nm to 100 nm,becoming thick (however, still in the nanometer regime) with age anddisease (reference is made to Parsons J R, “Cartilage,” In: Black J,Hastings G, editors, Handbook of Biomaterial Properties, Chapman andHall, London: 1998, 40, the disclosure of which is hereby incorporatedby reference herein). Articular cartilage has a limited capacity forrepair when the tissue is damaged or diseased (reference is made toMankin H J, et al., “Metabolism of Articular Cartilage,” In: Simon S P,et al., editors, Form and Function of Bone in Orthopaedic Basic Science,American Academy of Orthopaedic Surgeons, Columbus, Ohio: 1994, 12, thedisclosure of which is hereby incorporated by reference herein). Thislimited self-regeneration capability has made it difficult to createsuccessful cartilage-tissue engineered replacements.

SUMMARY

A substrate for promoting growth of chondrocytes to repair articularcartilage is disclosed. The substrate comprises a polymeric materialcomprising aligned and nano-sized surface structures.

The polymeric material comprises, for example, poly (lactic/glycolicacid) material. Aligned ridges are formed on the surface by stretchingthe poly (lactic/glycolic acid) material. The substrate is etched with acompound (e.g., NaOH) to form the nano-sized surface structures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-1 d show scanning electron micrographs of PLGA substrates;

FIG. 1 a shows non-aligned, conventional PLGA;

FIG. 1 b shows aligned, conventional PLGA;

FIG. 1 c shows non-aligned, nano-structured PLGA;

FIG. 1 d shows aligned, nano-structured PLGA;

FIG. 2 shows four-hour chondrocyte adhesion experiment results whereinchondrocyte adhesion is the greatest on the conventional substratescompared to nano dimensional substrates, seeding density was at 5,000cells/cm², values are mean +/− SEM, n=3, * p<0.05 (compared to celldensity on respective non-aligned PLGA), ** p<0.01 (compared to celldensity on aligned conventional substrate);

FIG. 3 shows 1, 3, and 6-day proliferation experiment results whereinchondrocytes in chondrocyte growth media were seeded (5,000 cells/cm²)onto reference glass and PLGA substrates as described in sectionMaterials and Methods and cultured for 1, 3, and 6 days, the number ofchondrocytes was higher on nanostructured PLGA, Compared to conventional(micron-sized) controls, nanostructured substrates have more affinity tochondrocytes, values are mean +/− SEM, n=3, * p<0.05 (compared to celldensity at day 1 on respective substrate), ** p<0.05 (compared to celldensity on conventional PLGA substrates), # p<0.1 (compared to celldensity at day 1 on respective substrate);

FIG. 4 shows increased migration distances on nano-structured andaligned PLGA wherein chondrocytes were allowed to adhere on thesubstrates of interest for 4 hours in the presence of TEFLON® inserts,TEFLON® fences were subsequently removed and cells were allowed tomigrate for 2, 4, and 6 days, migration distance across the surfaceswere determined at the end of each time period, and results are based ontwo separate experiments of triplicate samples;

FIG. 5 is a diagrammatic view showing a layer of PLGA formed on a layerof silastic film;

FIG. 6 is a diagrammatic view showing stretching of the PLGA to formaligned ridges on the surface of the PLGA; and

FIG. 7 is a diagrammatic view showing treatment of the PLGA with acompound such as NaOH to form nano-sized structures on the surface ofthe PLGA.

DETAILED DESCRIPTION

In the present study, bioresorbable poly (lactic/glycolic acid) (PLGA)materials with modified surface characteristics were tested as scaffoldsto enhance chondrocyte (cartilage-synthesizing cells) adhesion andproliferation. Nano-dimensional features were aligned on the PLGAsurfaces to simulate the physiological structure of articular cartilage.Nanostructured PLGA topographical features were created by etching thesurface with 10N NaOH for 1 hour. Alignment was created by mechanicallystretching the etched PLGA longitudinally at 60% strain while curing.The results showed decreased chondrocyte numbers on nano-dimensionalsubstrates after 4-hour adhesion experiments. However, higher celldensities were observed on nano-dimensional substrates after 1, 3, and 6days during proliferation experiments, indicating for the first timethat chondrocytes proliferated at a faster rate on the nanostructuredsubstrates. Furthermore, migration studies using TEFLON® (i.e.,polytetraflouroethylene) fences demonstrated longer distances ofpreferential alignment of chondrocytes along aligned nanostructured PLGAridges after 2, 4, and 6 days. The present study thus provided the firstevidence that mimicking the topographical structure of articularcartilage by aligning nanometer surface features on PLGA will enhancechondrocyte proliferation needed for articular cartilage restoration.

The overall objective of this study was to develop a biodegradableimplant that mimics collagen structure and dimension to enhance theadhesion and growth of chondrocytes and thus promote regeneration ofcartilage. More specifically, inducing alignment of surface features onscaffold materials coupled with increasing nano-dimensional surfaceroughness may simulate the environment chondrocytes experience in situ.In this study, chondrocyte adhesion, proliferation, and migrationdistances were determined on aligned nanostructuredpoly(lactic-co-glycolic acid) (PLGA) substrates.

MATERIALS AND METHODS

Substrate Preparation

Poly (lactic/glycolic acid) (PLGA; 50:50 wt %; Polysciences, Inc.)copolymer films were synthesized using chloroform and heat treatmentaccording to standard techniques [reference is made to Mikos A, ThorsenA, Czerwonka L, Boa Y, Winslow D, Vancanti J, and Langer R, “Preparationand Characterization of Poly(L-lactic) Foams for Cell Transplantation,”,Polymer 1990; 35:1068, the disclosure of which is hereby incorporated byreference herein]. The copolymer solution (0.25 g PLGA in 2 mLchloroform) was poured onto a silastic film (7 cm×3.5 cm×1 mm), assuggested, for example, in FIG. 5, and strained by 60% using clamps, assuggested, for example, in FIG. 6; this induced aligned ridges on thePLGA surface. The copolymer assembly was covered with parafilm;air-dried overnight at room temperature and placed in a vacuum (at 15-inHg pressure) for 48 hours to allow chloroform to evaporate. Samples ofPLGA (1 cm×0.5 cm×0.05 cm) were cut from the bulk polymer film for usein all experiments. Some polymer scaffolds of PLGA were soaked for 1hour in 10N NaOH at room temperature to create nano-structured (surfacefeatures less than 100 nm) substrates, as suggested, for example, inFIG. 7. Unmodified PLGA (processed the same way except for NaOHtreatment) served as conventional (i.e., control) PLGA substrates.Borosilicate glass coverslips etched with 1N NaOH, sterilized byautoclaving, were used as reference substrates.

Surface Characterization

Surface topography and roughness of the substrates were evaluated usingscanning electron microscopy (JOEL JSM-840). Samples were coated withgold via a sputter-coater at ambient temperature. Micrographs were takenat 250× with 5-7 kV.

Cytocompatibility

Human chondrocytes (PN=6-12; Cell Applications, Inc.) were culturedusing chondrocyte growth media (Cell Applications, Inc.) under standardcell culture conditions (i.e., a 37° C., humidified, 5% CO₂/95% airenvironment). For proliferation experiments, human chondrocytes wereseeded at a density of 5,000 cells/cm² onto each substrate and incubatedunder standard cell culture conditions in chondrocyte growth media for1, 3 and 6 days. Chondrocyte growth media was replaced every other day.Adhesion experiments were performed under similar conditions asproliferation, except cells were cultured for only 4 hours. At the endof each time period, the cells were fixed with 4% formaldehyde andstained with Coomassie Blue. The cells were counted at five randomfields on each substrate using a brightfield light microscope, and thesenumbers were averaged and reported as cell density or cells/cm². Allexperiments were done in duplicate, and repeated at least three separatetimes.

Chondrocyte migration was investigated on the substrates of interest todetermine whether the surface features on the substrates would influencechondrocytes to align as they migrate. TEFLON® inserts with rectangularwell (0.6 cm×0.3 cm), off center from the insert, were employed to trapthe cells and to give them a starting line for migration. These insertsand 2 mL chondrocyte growth media were placed into the 12-well plate,and 50,000 chondrocytes were micropipetted into the small well createdby the TEFLON® insert. The TEFLON® fences covered the entire substratesurface except for a rectangular area. Cells were allowed to adhere for4 hours, and the inserts were removed carefully. Free-floating cellswere aspirated off and the media was replaced. Then the cells were freeto migrate for 2, 4, and 6 days under standard cell culture conditions.Growth media was replaced every other day. After each incubation period,cells were fixed with 3.7% formaldehyde, stained with Coomassie Blue andmigration distance determined by light microscopy at 10×.

Statistical Analysis

Data were analyzed using standard Student t tests with p<0.05 indicatingstatistical significance.

RESULTS

Substrate Characterization

Scanning electron microscope (SEM) images provided evidence that surfacefeatures were aligned (indicated by arrows) by using strain (FIG. 1 bcompared to FIG. 1 a and FIG. 1 d compared to FIG. 1 c). Moreover,treating PLGA with NaOH created nanostructured surface features (FIG. 1c compared to FIG. 1 a and FIG. 1 d compared to FIG. 1 b).

Chondrocyte Interaction with Substrates

The present study showed the largest number of chondrocytes on theconventional substrates after 4-hour adhesion experiments, which is incontrast to a previous study [reference is made to Kay S, Thapa A,Haberstroh K M, and Webster T J, “Nanostructured Polymer/NanophaseCeramic Composites Enhance Osteoblast and Chondrocyte Adhesion,” TissueEngineering, in press, 2002, which is hereby incorporated by referenceherein]. This may be due to differences in population numbers or seedingdensity, since the previous study was done at lower seeding density andlower population numbers (1-5). As for the differences in aligned versusnon-aligned substrates, more chondrocytes adhered to the alignedsubstrates compared to the non-aligned samples as shown in FIG. 2.

The results of the 1, 3, and 6-day proliferation experiments indicatedthat the chondrocyte proliferation rate increased on nanostructuredsubstrates compared to the conventional samples. FIG. 3 displays theincreased cell numbers on nanostructured samples. In contrast to theadhesion data, significant cell count differences were not seen betweenaligned and non-aligned substrates.

Chondrocyte migration study was performed to determine the distance thecells spread for 2, 4, and 6 days on each of the substrates previouslydescribed. The results are displayed in FIG. 4 below. The longerdistances were obtained on nanostructured versus conventional PLGA, andthe same trend was observed on aligned versus nonaligned substrates.

DISCUSSION AND CONCLUSIONS

Adhesion results from the present study demonstrated lower initial celldensity on nano-dimensional substrates. However, increasing surfaceroughness (FIG. 1) enhanced the proliferation of chondrocytes accordingto the present study (FIG. 3). This indicates that the proliferationrate is higher on nanostructured substrates compared to the conventionalsamples, which implies the possibility of enhanced cartilageregeneration. This is further evidenced by the migration distances ofchondrocytes from the 2, 4, and 6-day experiments which showed thatchondrocytes tend to migrate faster on aligned and nanostructuredsubstrates (FIG. 4).

In proliferation experiments, no difference in cell counts was observedbetween cells on aligned and on non-aligned substrates. However, in thepresence of grooves or local alignment, chondrocytes grew along thedirection of alignment (reference is made to Park G E, Savaiano J K,Park K, Webster T J, “An In Vitro Study of Chondrocyte Function onNanostructured Polymer/Ceramic Formulations to Improve CartilageRepair,” NANO2002 Conference Abstract Book, Orlando, Fla., pg. 269,2002, the disclosure of which is hereby incorporated by referenceherein). One possible explanation for this phenomenon is that due to thepresence of grooves and protrusion on the surface, proteins from thecell culture media interacted more favorably with alignednano-structures to promote cell spreading. Since proteins arenanostructured (most <100 nm), their adsorption and conformation may beinfluenced to a larger degree on surfaces with nanometer (<100 nm)compared to conventional features. Another reason may be the chemicalchanges on the surface due to the etching process. However, previousstudies have shown that when isolating surface topography from chemicaletching changes in PLGA, bladder and vascular cells prefer thenanostructured compared to the conventional surface features (referenceis made to Thapa A, Webster T J, and Haberstroh K M, “An Investigationof Nano-Structured Polymers for Use as Bladder Tissue ReplacementConstructs,” Materials Research Society Symposium Proceedings711:GG3.4.1-GG3.4.6, 2002, and is made to Miller D M, Thapa A,Haberstroh K M, and Webster T J, “An In Vitro Study of Nano-FiberPolymers for Guided Vascular Regeneration,” Materials Research SocietySymposium Proceedings 711:GG3.2.1-GG3.2.4, 2002, the disclosures ofwhich are hereby incorporated by reference herein). Therefore, it isreasonable to speculate that alignment of nanostructured PLGA surfacefeatures alone serves to spatially control chondrocyte proliferation.The fact that the effect is long-term suggests that the cells can anchorwell due to mechanical interlocking through the protein layer that maymore tightly adsorb to the nanometer rough surface.

In summary, the results of the present study have shown a potential formodifying biodegradable polymer surface characteristics by creatingaligned, nanometer features to enhance chondrocyte growth for articularcartilage repair.

1. A substrate for promoting growth of chondrocytes to repair articularcartilage, the substrate comprising a polymeric material, wherein saidpolymeric material comprises aligned and nano-sized surface structuresformed on the surface of said polymeric material of said substrate, saidnano-sized surface structures comprising grooves and protrusions,wherein the grooves have a maximum depth, and the protrusions have amaximum height, of less than 100 nm.
 2. The substrate of claim 1,wherein the polymeric material comprises poly(lactic/glycolic acid). 3.The substrate of claim 1, wherein the polymeric material isbiodegradable.
 4. The substrate of claim 1, wherein the polymericmaterial comprises a polymeric film.
 5. The substrate of claim 1,wherein the polymeric material comprises a biodegradablepoly(lactic/glycolic acid) film.
 6. The substrate of claim 1, comprisinga population of chondrocytes introduced on the surface of the polymericmaterial.
 7. The substrate of claim 1, comprising chondrocytes grownalong the aligned surface structures.
 8. A substrate comprising apolymeric material with nano-sized surface structures, wherein adimension of the nano-sized surface structures is less than 100 nm, saiddimension selected from the group consisting of cross-sectional diameterand height.
 9. The substrate of claim 8, wherein the surface structurescomprise aligned grooves and protrusions.
 10. A substrate comprising apolymeric material wherein the surface of said polymeric materialcomprises nano-sized protrusions and grooves, wherein a dimension ofsaid protrusions and grooves is less than 100 nm.
 11. The substrate ofclaim 10, wherein the polymeric material surface comprises abiodegradable poly(lactic/glycolic acid) film, said film furthercomprising a population of chondrocytes introduced on the biodegradablepoly (lactic/glycolic acid) film.
 12. The substrate of claim 10 whereinthe nano-sized protrusions and grooves are aligned.